The present invention relates to immunology and, in particular, to immunization of hosts using nucleic acid to provide protection against infection by Chlaymdia.
DNA immunization is an approach for generating protective immunity against infectious diseases (ref. 1xe2x80x94throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure) Unlike protein or peptide based subunit vaccines, DNA immunization provides protective immunity through expression of foreign proteins by host cells, thus allowing the presentation of antigen to the immune system in a manner more analogous to that which occurs during infection with viruses or intracellular pathogens (ref. 2). Although considerable interest has been generated by this technique, successful immunity has been most consistently induced by DNA immunization for viral diseases (ref. 3). Results have been more variable with non-viral pathogens which may reflect differences in the nature of the pathogens, in the immunizing antigens chosen, and in the routes of immunization (ref. 4). Further development of DNA vaccination will depend on elucidating the underlying immunological mechanisms and broadening its application to other infectious diseases for which existing strategies of vaccine development have failed.
Chlamydia trachomatis is an obligate intracellular bacterial pathogen which usually remains localized to mucosal epithelial surfaces of the human host. Chlamydiae are dimorphic bacteria with an extracellular spore-like transmission cell termed the elementary body (EB) and an intracellular replicative cell termed the reticulate body (ref. 5). From a public health perspective, chlamydial infections are of great importance because they are significant causes of infertility, blindness and are a prevalent co-factor facilitating the transmission of human immunodeficiency virus type 1 (ref. 6). Protective immunity to C. trachomatis is effected through cytokines released by Th1-like CD 4 lymphocyte responses and by local antibody in mucosal secretions and is believed to be primarily directed to the major outer membrane protein (MOMP), which is quantitatively the dominant surface protein on the chlamydial bacterial cell and has a molecular mass of about 40 kDa (ref. 19).
Initial efforts in developing a chlamydial vaccine were based on parenteral immunization with the whole bacterial cell. Although this approach met with success in human trials, it was limited because protection was short-lived, partial and vaccination may exacerbate disease during subsequent infection episodes possibly due to pathological reactions to certain chlamydial antigens (ref. 8). More recent attempts at chlamydial vaccine design have been based on a subunit design using MOMP protein or peptides. These subunit vaccines have also generally failed, perhaps because the immunogens do not induce protective cellular and humoral immune responses recalled by native epitopes on the organism (ref. 9).
EP 192033 describes the provision of DNA construct for the expression, in vitro, of Chlamydia trachomatis MOMP polypeptides comprising the following operably linked elements:
a transcriptional promoter,
a DNA molecule encoding a C. trachomatis MOMP polypeptide comprising a MOMP polynucleotide at least 27 base pairs in length from a sequence provided in Appendix A thereto, and
a transcriptional terminator, wherein at least one of the transcriptional regulatory elements is not derived from Chlamydia trachomatis. There is no disclosure or suggestion in this prior art to effect DNA immunization with any such constructs.
WO 94/26900 describes the provision of hybrid picornaviruses which express chlamydial epitopes from MOMP of Chlamydia trachomatis and which is capable of inducing antibodies immuno-reactive with at least three different Chlamydia serovars. The hybrid picornavirus preferably is a hybrid polio virus which is attenuated for human administration.
The present invention is concerned with nucleic acid immunization, specifically DNA immunization, to generate in a host protective antibodies to a MOMP of a strain of Chlamydia. DNA immunization induces a broad spectrum of immune responses including Th1-like CD4 responses and mucosal immunity.
Accordingly, in one aspect, the present invention provides an immunogenic composition for in vivo administration to a host for the generation in the host of a protective immune response to a major outer membrane protein (MOMP) of a strain of Chlamydia, comprising a non-replicating vector comprising a nucleotide sequence encoding a MOMP or MOMP fragment that generates a MOMP-specific immune response, and a promoter sequence operatively coupled to the nucleotide sequence for expression of the MOMP or MOMP fragment in the host; and a pharmaceutically-acceptable carrier therefor.
The nucleotide sequence may encode a full-length MOMP protein or may encode a fragment, such as the N-terminal half of MOMP or a fragment that encompasses epitopic sequences. The nucleotide sequence may encode a MOMP or MOMP fragment which stimulates a recall immune response following exposure to wild-type Chlamydia. The promoter may be the cytomegalovirus promoter.
The fragment that encompasses epitopic sequences may include one or more conserved domain (CD) sequences and/or one or more variable domain (VD) sequences of MOMP from a strain of Chlamydia. In particular, the fragment may encompass the CD2 and VD2 sequences, CD3 and VD3 sequences and CD5 sequence. Clones containing nucleotide sequences encoding such fragments are termed clones CV2, CV3 and CD5 herein. Clones CV2 encompasses nucleotides 247 to 468 of Chlamydia trachomatis MOMP gene, clone CV3 encompasses nucleotides 469 to 696 of Chlamydia trachomatis MOMP gene and clone CV5 encompasses nucleotides 931 to 1098 of Chlamydia trachomatis MOMP gene. Non-replicating vectors comprising such sequences are novel and constitute further aspects of the invention.
Accordingly, in an additional aspect of the invention, there is provided a non-replicating vector, comprising a nucleotide sequence encoding a region comprising at least one of the conserved domains 2, 3 and 5 of a major outer membrane protein of a strain of Chlamydia, and a promoter sequence operatively coupled to the nucleotide sequence for expression of the at least one conserved domain in a host. In this aspect of the invention, the various options and alternatives discussed above and below may be employed.
The strain of Chlamydia may be a strain of Chlamydia inducing chlamydial infection of the lung, including Chlamydia trachomatis or Chlamydia pneumoniae. The non-replicating vector may be plasmid pcDNA3 into which the nucleotide sequence is inserted. The immune response which is stimulated may be predominantly a cellular immune response.
In a further aspect of the invention, there is provided as a method of immunizing a host against disease caused by infection with a strain of Chlamydia, which comprises administering to the host an effective amount of a non-replicating vector comprising a nucleotide sequence encoding a major outer membrane protein (MOMP) of a strain of Chlamydia or a MOMP fragment that generates a MOMP-specific immune response, and a promoter sequence operatively coupled to the nucleotide sequence for expression of the MOMP or MOMP fragment in the host.
In this aspect of the present invention, the various options and alternatives discussed above may be employed.
The non-replicating vector may be administrated to the host, including a human host, in any convenient manner, such as intramuscularly or intranasally. Intranasal administration stimulated the strongest immune response in experiments conducted herein.
The present invention also includes, in an additional aspect thereof, a method of using a gene encoding a major outer membrane protein (MOMP) of a strain of Chlamydia or MOMP fragment that generates a MOMP-specific immune response, to produce an immune response in a host, which comprises isolating the gene, operatively linking the gene to at least one control sequence to produce a non-replicating vector, the control sequence directing expression of the MOMP or MOMP fragment when introduced into a host to produce an immune response to the MOMP or MOMP fragment, and introducing the vector into a host.
A further aspect of the present invention provides a method of producing a vaccine for protection of a host against disease caused by infection with a strain of Chlamydia, which comprises isolating a nucleotide sequence encoding a major outer membrane protein (MOMP) of a strain of Chlamydia or a MOMP fragment that generates a MOMP-specific immune response, operatively linking the nucleotide sequence to at least one control sequence to produce a non-replicating vector, the control sequence directing expression of the MOMP or MOMP fragment when introduced to a host to produce an immune response to the MOMP or MOMP fragment, and formulating the vector as a vaccine for in vivo administration to a host. The invention extends to the vaccine produced by this method.
Advantages of the present invention, therefore, include a method of obtaining a protective immune response to infection carried by a strain of Chlamydia by nucleic acid immunization of nuelcic acid sequence encoding the major outer membrane protein of a strain of Chlamydia or a fragment of the outer membrane protein that generates a MOMP-specific immune response.